Western blotting (or, protein immunoblotting) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate, cell lysate or other protein containing samples. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein. For certain applications, the proteins are probed in the gel without a transfer step. However, further description of western blot methodology is based on protein samples transferred to a membrane.
During the detection process the membrane is probed for the protein of interest by the use of an antibody specific for the protein of interest. Due to possibilities of increased signal amplification and to avoid negative effects on target specific affinity related to primary antibody conjugation, this traditionally takes place in a two-step process (using a primary target specific antibody and a secondary labeled antibody specific for the primary antibody), although there are now one-step detection methods available for certain applications. The one-step method allows the process to occur faster and with a lower amount of consumables, but sensitivity and specificity may be compromised. This requires a probe antibody which both recognizes the protein of interest and contains a detectable label, probes which are often available for known protein tags. The primary probe is incubated with the membrane in a manner similar to that for the primary antibody in a two-step process, and is then ready for direct detection after a series of wash steps.
Preparing or raising antibodies against a partly purified target protein or proteins with weak immunogenic properties may result in low immuno-specificity and antibodies from suppliers are not always of sufficiently high quality. Affinity purification could be used to overcome the problem, but requires purified target protein and involves risks with sample handling including e.g. loss of antibody, activity and stability issues. Improper storage and aged antibodies can give similar reduced detection performance. When a protein target (mw size may be unknown) is lacking specific antibodies of high quality, the true immunogenic protein band corresponding to the target is very difficult to determine in a conventional Western blotting experiment.
For many target proteins there is a limited availability of highly specific antibodies, resulting in a Western blot assay with low specificity. This may lead to that it is impossible to identify correct band when molecular weight of target protein is unknown. Also there is a risk of identifying the wrong band if unspecific bands are located close to the specific band, particularly if the unspecific bands are prominent. This may be a problem even when the molecular weight of the target protein is known.
Thus, there is a need of improved methods for increasing the specificity in the identification of target proteins especially in Western blotting assay formats.